Frank van der Meer

Induction of Toll-like receptor 4 signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way.

LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in avian macrophages and observe whether TLR4 mediated induction of NO can elicit antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQ-NCSU cells were treated with LPS and the resultant supernatant was then transferred to ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a selective inhibitor, S-methylisothiourea sulfate that inhibits inducible NO synthase. This observation confirms that the antiviral activity is positively correlated with NO production. The data show that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens that require further evaluation in vivo.

The modification and evaluation of an ELISA test for the surveillance of Mycobacterium avium subsp. paratuberculosis infection in wild ruminants.

BackgroundEnzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit) for use with species for which it was not originally developed: elk (Cervus elaphus), bison (Bison bison) and caribou (Rangifer tarandus). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species.ResultsWe found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively.ConclusionsDue to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were developed. In order to generate reliable test results, it is essential to evaluate the test and perform modifications if deemed necessary. Despite the challenges inherent to wildlife diagnostics, we have shown that several methods can be used to improve confidence in test results.

Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot

Bovine respiratory disease complex (BRDc) is a major cause of morbidity and mortality in beef cattle. There is recent evidence suggesting that the nasopharyngeal microbiota has a key role in respiratory health and disease susceptibility in cattle. However, there is a paucity of knowledge regarding evolution of the nasopharyngeal microbiota when cattle are most likely to develop BRDc (i.e., from weaning to 40days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40days after arrival at a feedlot. Deep nasal swabs (DNS) from 30 Angus-cross steers were collected at weaning, on arrival at a feedlot, and at day 40 after arrival. The DNA was extracted from DNS and the hypervariable region V3 of the 16S rRNA gene was amplified and sequenced (Illumina MiSeq platform). Nasopharyngeal microbiota underwent a profound evolution from weaning to arrival at the feedlot and from arrival to day 40, with the abundance of 92 Operational Taxonomic Units (OTUs) significantly changing over time. Mycoplasma (M. dispar and M. bovirhinis) was the most abundant genus in the nasopharynx, accounting for 53% of the total bacterial population. Because an evolving bacterial community may be less capable of resisting colonization by pathogenic bacteria, the instability of the nasopharyngeal microbiota documented in this study might explain why cattle are most likely to be affected with BRDc during the first weeks after weaning and arrival at a feedlot.

Pathogens at the livestock-wildlife interface in Western Alberta: does transmission route matter?

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk☆

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1-V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk.

Contrasting Results of Culture-Dependent and Molecular Analyses of Mycobacterium avium subsp. paratuberculosis from Wood Bison

Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.

Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle

Bovine viral diarrhea virus (BVDV), a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI) cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

Compartmental HBV evolution and replication in liver and extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers

BACKGROUND Hepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence. OBJECTIVES To investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy. STUDY DESIGN A total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N=7 baseline, N=5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver. RESULTS Compare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2-0.08 copies/hepatocyte, p>0.05) or in PBMC 0.003-0.02 copies/PBMC, p>0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma. CONCLUSION Continuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy.

Contagious Ecthyma, Rangiferine Brucellosis, and Lungworm Infection in a Muskox (Ovibos moschatus) from the Canadian Arctic, 2014

Abstract An adult male muskox (Ovibos moschatus), harvested on 26 August 2014 on Victoria Island, Nunavut, in the Canadian Arctic, had proliferative dermatitis on the muzzle and fetlocks suggestive of contagious ecthyma or orf (Parapoxvirus). Histopathologic features of the lesions were consistent with this diagnosis. Orf virus DNA, phylogenetically similar to an isolate from a captive muskox of the Minnesota Zoo, US, was detected in the lesions by PCR using Parapoxvirus primers. Additionally, there was a metaphyseal abscess with a cortical fistula in the right metacarpus from which Brucella suis biovar 4 was isolated and identification supported by PCR. Brucella spp. antibodies were detected in serum. Finally, 212 nodules were dissected from the lungs. Fecal analysis and lung examination demonstrated co-infection with the lungworms Umingmakstrongylus pallikuukensis and Varestrongylus eleguneniensis. The zoonotic potential of orf and rangiferine brucellosis adds an important public health dimension to this case, particularly given that muskoxen are a valuable source of food for Arctic residents. Careful examination of these pathogens at a population level is needed as they may contribute to muskox population decline and potentially constitute a driver of food insecurity for local communities. This case underscores the importance of wildlife health surveillance as a management tool to conserve wildlife populations and maintain food security in subsistence-oriented communities.

Bacterial Genomics Reveal the Complex Epidemiology of an Emerging Pathogen in Arctic and Boreal Ungulates.

Northern ecosystems are currently experiencing unprecedented ecological change, largely driven by a rapidly changing climate. Pathogen range expansion, and emergence and altered patterns of infectious disease, are increasingly reported in wildlife at high latitudes. Understanding the causes and consequences of shifting pathogen diversity and host-pathogen interactions in these ecosystems is important for wildlife conservation, and for indigenous populations that depend on wildlife. Among the key questions are whether disease events are associated with endemic or recently introduced pathogens, and whether emerging strains are spreading throughout the region. In this study, we used a phylogenomic approach to address these questions of pathogen endemicity and spread for Erysipelothrix rhusiopathiae, an opportunistic multi-host bacterial pathogen associated with recent mortalities in arctic and boreal ungulate populations in North America. We isolated E. rhusiopathiae from carcasses associated with large-scale die-offs of muskoxen in the Canadian Arctic Archipelago, and from contemporaneous mortality events and/or population declines among muskoxen in northwestern Alaska and caribou and moose in western Canada. Bacterial genomic diversity differed markedly among these locations; minimal divergence was present among isolates from muskoxen in the Canadian Arctic, while in caribou and moose populations, strains from highly divergent clades were isolated from the same location, or even from within a single carcass. These results indicate that mortalities among northern ungulates are not associated with a single emerging strain of E. rhusiopathiae, and that alternate hypotheses need to be explored. Our study illustrates the value and limitations of bacterial genomic data for discriminating between ecological hypotheses of disease emergence, and highlights the importance of studying emerging pathogens within the broader context of environmental and host factors.